ES2208773T3 - APOPTOSIS CAUSED BY ANTI-HER2 MONOCLONAL ANTIBODY. - Google Patents

Abstract

THIS INVENTION DESCRIBES ANTI-HER2 ANTIBODIES, WHICH INDUCE APOPTOSIS IN CELLS THAT EXPRESS HER2. SUCH ANTIBODIES ARE USED TO "MARK" TUMORS WITH SUPER EXPRESSION OF HER2, FOR THEIR ELIMINATION BY THE IMMUNE SYSTEM OF THE GUEST. Likewise, CELLULAR HYBRIDOMA LINES THAT PRODUCE SUCH ANTIBODIES, CANCER TREATMENT METHODS USING SUCH ANTIBODIES AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM ARE DESCRIBED.

Description

Apoptosis caused by the monoclonal antibody anti-Her2.

Field of the Invention

The present invention relates to anti-Her2 antibodies and, more specifically, with anti-Her2 antibodies that induce apoptosis in cells expressing Her2.

Background of the invention

The Her2 oncogene encodes a membrane-associated glycoprotein referred to as p185HER-2 having tyrosine kinase activity. Her2 is a member of the subfamily of epidermal growth factor (EGF) receptors, which includes the EGF receptor and Her3 and Her4 receptors (Kraus et al., Proc. Natl. Acad. Sci. USA 86 , 9193-9197 (1989); Plowman et al., Proc. Nat. Acad. Sci. USA 90 , 1746-1750 (1993)). The Her2 sequence was described by Semba et al. (Proc. Natl. Acad. Sci. USA 82 , 6497-6501 (1985)); Coussens et al. (Science 230 , 974-976 (1985)). Schecter et al. described a related rat gene (Nature 312 , 515-516 (1984)).

Greater expression of the Her2 oncogene has been described in tumor cells and cell lines by several groups (Coussens et al., Cited above; King et al., Cited above). The greater expression of Her2 occurs as a result of gene amplification or greater expression of the single copy gene. These observations suggested that Her2 can be overexpressed in human cancer tissue. Slamon et al. (Slamon et al., Science 235 , 177-182 (1987); Slamon et al., Science 244 , 707-712 (1989)) examined Her2 expression levels in tumors taken from a large sample of patients of breast and ovarian cancer. It was found that almost 30% of these patients had amplification and overexpression of the Her2 gene, which was associated with a poor clinical outcome (increased recurrences and low survival rate), particularly in patients with positive breast cancer in nodules. The correlations given by Slamon have been confirmed in a series of studies (see, for example, Ro et al., Cancer Res. 49 , 6941-6944 (1989); Walker et al., J. Cancer 60 , 426-429 ( 1989); Wright et al., Cancer Res. 49 , 2087-2090 (1989); Berchuck et al., Cancer Res. 50 , 4087-4091 (1990); Kallioniemi et al., Int. J. Cancer 49 , 650 -655 (1991); Rilke et al., Int. J. Cancer 49 , 44-49 (1991)).

The presence of certain factors, such as the overexpression of Her2, which are indicative of a poor prognosis, may suggest that adjuvant therapy is appropriate after surgical excision. Adjuvant therapy may include high dose chemotherapy and autologous bone marrow transplantation. It has been recently described (Muss et al., N. Engl. J. Med. 330 , 1260-1266 (1994)) that breast cancer patients who have tumors exhibiting overexpression of Her2 enjoyed significant benefits thanks to therapy adjuvant

By analogy with other receptor tyrosine kinase proteins, it is assumed that a ligand for Her2 stimulates receptor phosphorylation. It has been described that a number of polypeptide factors increase the phosphorylation of tyrosine from Her2 and were assumed to be a ligand (Wen et al., Cell 64 , 559-572 (1992); Holmes et al., Science 256 , 1205- 1210; Marchionni et al., Nature 362 , 312-318 (1993); Falls et al., Cell 72 , 801-815 (1993)). However, there is no evidence that any of these factors is a true ligand that binds directly to Her2 and stimulates receptor phosphorylation. One approach to save the presence of ligand is to generate a monoclonal antibody ("mAb") of ligand type. Several groups have generated anti-Her2 mAbs using a Her2 cell surface receptor or a purified extracellular domain of the Her2 receptor (Yarden, Proc. Natl. Acad. Sci. USA 87 , 2569-2573 (1990); Hanwerth et al., Br. J. Cancer 68 , 1140-1145 (1993); Srinivas et al., Cancer Immunol. Immunother. 36 , 397-402 (1993); Stancovaski et al., Proc. Natl. Acad. Sci. USA 88 , 8691 -8695 (1991)). These mAbs stimulated phosphorylation of Her2 tyrosine from cells with overexpression, but were not fully characterized in terms of binding to, and phosphorylation of, each of Her2, Her3 or Her4 or in terms of kinase activation in transfected cells. with Her2.

The inhibitory effects of the growth of anti-Her2 mAbs on breast cancer cells have been previously described (Tagliabue et al., Int. J. Cancer 47 , 933-937 (1991); Hudziak et al., Mol. Cell. Biol. 9, 1165-1172 (1989); Drevin et al., Oncogen 2, 387-394 (1988); Fendly et al., Cancer Res. 50 , 1550-1558 (1990), Hanwerth et al., Cited above ; see also the review by Vitetta and Uhr, Cancer Res. 54 , 5301-5309 (1994)), but these effects were interpreted as cytostatic, since the removal of the antibody allowed to resume cell growth. Xu et al. (Int. J. Cancer 53 , 401-408 (1993)) described anti-Her2 antibodies that were cytotoxic for the growth of anchor independent tumor cells.

An anti-EGF receptor mAb was described that induced apoptosis in the human colorectal carcinoma cell line, DiFi, that overexpresses the EGF receptor, and that induced morphological changes at concentrations of 5 to 20 nM. These effects were interpreted in terms of blocking the binding of EGF to the related receptor by competitive mAb and lack of mitogenic activity of mAb (Wu et al., J. Clin. Invest. 95 , 1897-1905 (1995)) .

Apoptosis, or programmed cell death, is a form of cell death characterized by cell shrinkage and DNA fragmentation. Collapse of the cell nucleus is apparent, since chromatin is fragmented into single or multiple mononucleosomal units, a process mediated by an endogenous endonuclease. Apoptosis is distinct from necrotic cell death, which results in cell swelling and release of intracellular components (Kerr et al., Br. J. Cancer 26 , 239-257 (1972); Wyllie et al., Int. Rev Cytol. 68 , 251-306 (1980); Wyllie, Nature 284 , 555-556 (1980)). Apoptotic cells, without releasing these components, are phagocytosed and therefore degraded (Savill et al., Nature 343 , 170-173 (1990)). Therefore, apoptosis results in an efficient process for the elimination of non-viable cells by the host's own defense mechanisms.

Deshane, J. et al. ("Intracellular antibody knockout of the erbB2 oncoprotein achieves targeted eradication of tumor targets by induction of apoptosis ", J. Invest. Med., Vol. 32, No. Suppl. 2, April 1995, page 328A) refer to the suppression by intracellular antibodies of oncoprotein erbB-2 and targeted eradication of targets Tumor by induction of apoptosis in this process. Deshane, J. and cabbage. use gene constructs designed to code single chain immunoglobulins (sFvs) with specificity anti-ErbB-2 for expression intracellular antibody anti-erbB-2.

Grim, J. et al. ("Induction of apoptotic cell death in erbB-2 overexpressing tumor cells of diverse histologic subtypes mediated by intracellular localization of an anti-erbB-2 sFv ", Cancer Gene Therapy, Vol. 1, No. 4, December 1994, pages 333-334) refer to the induction of death apoptotic cell in overexpressing tumor cells erbB-2 of various histological mediated subtypes by the intracellular location of an SSV anti-erbB-2. Gene construction de Grim, J. et al. is administered to the carcinoma cell line of human ovary SKOV3 by the method of adenovirus-polylysine ('' AdpL) and expression intracellular single chain antibody anti-erbB-2 leads to regulation decreasing expression of erbB-2 of the cell surface and induction of apoptosis in cells Tumor

Curiel, O. ("Strategies to accomplish targeted tumor cell cytotoxicity ", Gene Therapy, Vol. 2, No. Suppl. 1, November 1995, page 520) refers to strategies for achieve the cytotoxicity of the intended tumor cells. Curiel, O. Describes the intracellular expression of an antibody of single chain (sFv) anti-erbB-2 sFvs and the induction of apoptosis.

WO 94/00136 A (January 6, 1994) relates with the use of a combination of monoclonal antibodies anti-erbB-2 for prevention and treatment of human malignancies by induction of apoptosis.

Kita, Y. et al. ("ErbB receiver activation, cell morphology changes and apoptosis induced by anti-Her2 monoclonal antibodies ", Bioch. Biophys. Res. Comm., Vol. 226, No. 1, September 4, 1996, pages 59-69) refer to the activation of receptors erbB, changes in cell morphology and induced apoptosis by anti-Her2 monoclonal antibodies.

WO 96/07321 A (March 14, 1996) refers to methods to modulate protein function in cells using intracellular antibody homologs.

It is an object of the invention to generate antibodies for Her2 that induce apoptosis in cells expressing Her2 and of this mode "mark" said cells for removal of the Guest. Antibodies are useful for inducing apoptosis in tumors This represents a substantial improvement with regarding the antibody therapy currently available for the cancer, which typically leads to the death of tumor cells by antibodies together with a cytotoxic agent. The agents Cytotoxic generally produce unwanted side effects, which, if serious, can lead to a reduction or interruption of treatment This approach allows the death of tumor cells by the host's immune system, thus avoiding the effects of cytotoxic agents and cell necrosis Tumor induced by such agents.

Summary of the Invention

An embodiment of the present invention consists of in an anti-Her2 antibody or fragment thereof that induces apoptosis in cells expressing Her2. It has been seen that a antibody that stimulates phosphorylation of Her2 receptors in cell lines also has the unexpected effect of inducing changes in cells expressing Her2 characteristic of the apoptosis These changes include DNA fragmentation and loss of viability and are observed in the population of treated cells in 24 hours Said antibody is useful for labeling cells that overexpress Her2 for elimination by the mechanisms of guest defense.

In a preferred embodiment, the antibody before described recognizes an epitope of a Her2 polypeptide that is recognized by the monoclonal antibody produced by the line ATCC hybridoma cell No. HB-12078. Epitope was different from epitopes recognized by other antibodies that they also joined Her2, but they did not induce apoptosis, which suggests that the region of Her2 that interacts with the antibody is important to provoke an apoptotic response. Antibodies that induce apoptosis may exist as antibodies of length total that have variable and constant regions intact or regions thereof that retain the binding to Her2 and the apoptosis Antibodies can be produced by lines hybridoma cells or by recombinant DNA methods.

Preferably, the antibody described above is a monoclonal antibody and, more preferably, an antibody humanized An antibody is also preferred as described. Previously it is a human antibody.

Another embodiment of the present invention it consists of a hybridoma cell line capable of producing the antibody described above.

An antibody as described above is also a preferred embodiment of the present invention where the fragment is an F (ab) or Fab 'fragment. Preferably, the antibody described above is produced by the ATCC hybridoma cell line No. HB-12078 according to the present invention.

The present invention also provides the ATCC hybridoma cell line No. HB-12078 and an antibody produced by the ATCC hybridoma cell line No. HB-12078 .

Preferably, the cells expressing Her2 according to the present invention they are tumor cells. Plus preferably, tumor cells are derived from cancers mammary, ovarian, prostate, gastric and colorectal.

It is predicted that a series of cancers, including breast, ovarian, prostate and colorectal cancers are more invasive and therefore more lethal when they exhibit overexpression of Her2. The correlation between the expression of Her2 and a poor prognosis (more recurrences and higher mortality) in certain cancers has made Her2 an important goal for therapeutics of cancer

Another embodiment of the present invention is an in vitro method for inducing apoptosis in cells expressing Her2, consisting of administering an amount of the antibody described above sufficient to induce apoptosis. Preferably, the cells in the aforementioned in vitro method are cancer cells.

Another embodiment of the present invention is the use of an antibody as indicated above to prepare a medicine for the treatment of cancer. Preferably, the medication, which includes the antibody described above, induces apoptosis

Another embodiment of the present invention is a pharmaceutical composition containing an amount of an antibody of claim 1 sufficient to induce apoptosis in mixture with a pharmaceutically acceptable adjuvant. Preferably, the composition described above includes an antibody, which is a monoclonal antibody More preferably, the antibody is a humanized antibody A composition is also preferred as per has previously indicated where the antibody is an antibody human.

Description of the figures

Figure 1. Binding of mAb74 to glycosylated sHer2 and deglycosylated by Western blot analysis. (a) Degree of deglycosylation of Her2 by CHO staining after a SDS-PAGE non-reducing; (b) binding of mAb74 to Her glycosylated and deglycosylated as analyzed by Western blotting after a non-reducing SDS-PAGE.

Figure 2. Tyrosine phosphorylation of Her2 and Her3 induced by mAb stimulation in SKBR3. SKBR3 cells were seeded in a 48-well plate for 5 min. at 37 ° C for 18 hours before stimulation with mAb. Cells were solubilized with SDS sample buffer. The solubilized samples were electrophoresed in 6% polyacrylamide gels, followed by Western blotting and probing with anti-phosphotyrosine antibody. (a) All mAb concentrations were 250 nM in DMEM. A differentiation factor -? 2 nM neu (NDF?) Was used as a positive control. (b) Dose dependence of tyrosine phosphorylation mAb.

Figure 3. Inhibition by soluble Her2 receptor of receptor tyrosine phosphorylation induced by mAb. The phosphorylation assay is similar to that described in Figure 2. The cells were incubated with 250 nM mAb with different sHer2 concentrations.

Figure 4. Tyrosine phosphorylation of transfected cell line receptors, Her2 / 32D and HEG / 32D, induced by mAb stimulation. For the phosphorylation test, a cell pellet was obtained by centrifugation, washed with PBS and it was then incubated with 100 µL of 250 nM mAbs in RPMI for 5 min. at 37 ° C, followed by completion by adding 1 ml of PBS ice cream and centrifugation at 4 ° C. The supernatant was discarded and added SDS sample buffer to the centrifuged pellet. The Sample at 6% SDS-PAGE, followed by Western blotting and probing with anti-PTY Basal phosphorylated sample A431 was used as positive control

Figure 5. Cellular morphological change induced by mAbs Cells were grown (a-d, Her2 / MCF7; e, f, MDAMB453) with 1% FBS in culture medium with or without mAb. To the After 5 days, the cells were observed and photographed. (a, e) control (without mAb). (b) mAb74 250 nM. (c) mAb83 250 nM. (d) mAb42b 250 nM (f) mAb74 100 nM.

Figure 6. Detection of apoptotic cells with a Modified TUNEL method. MDAMB453 cells were incubated (a-d) or Her2 / MCF7 cells (e, f) with or without mAbs in culture medium with 1% FBS for one day, followed by a apoptosis assay. (a, e) control (without mAb). (b) mAb74 50 nM. (c, f) mAb74 500 nM. (d) mAb42b 500 nM.

Detailed description of the invention

Monoclonal antibodies (mAbs) have been generated which bind to Her2 by immunizing mice with purified soluble Her2. Soluble Her2 was expressed and purified as described in the Example 1. mAbs that bound soluble Her2 in Enzyme-linked immunosorbent assays (EIA) to cloning by dilution and new study by EIA and BIAcore for Her2 binding (Example 2). Ten clones were selected for further analysis. It was found that the purified antibodies of these clones bound preferentially to soluble Her2 and showed little or no binding to soluble Her3 and Her4. The biological effects of antibodies selected were studied in terms of dimerization of receptors, receptor phosphorylation and changes in the cellular physiology All antibodies studied formed 2: 1 complexes (receptor: antibody) with Her2 (Example 4). Three different antibodies stimulated phosphorylation of Her2 and Her3 receptors in SKBR3 cells and Her2 receptors, Her3 and Her4 in MDAMB453 cells. The phosphorylation of all receptors were inhibited by soluble Her2, suggesting that the ligand type effects of mAbs are directly mediated through Her2.

An antibody, mAb74, induced dramatic changes in the physiology of cells expressing Her2 (Examples 5 and 6). The treatment of MCF7 cells transfected with a Her2 gene from total length or treatment of MDAMB453 cells expressing Her2 naturally with mAb74 resulted in a marked change in cell morphology and extensive cell death. Other antibody, mAb83, showed a moderate effect on cell morphology. In non-viable cells, apoptosis had been induced, as evidenced by extensive DNA fragmentation. However, a subpopulation of cells escaped the activity of mAb74 and were not apoptotic

The invention provides an antibody or fragment thereof that induces apoptosis in cells that express Her2 As used herein, the term "apoptosis" refers to programmed cell death characterized by nuclear collapse and DNA degradation Cells that suffer apoptosis in response to  the antibodies of the invention will have at least Her2 in the cell surface and eventually Her3 and Her4. It is preferred that the tackled cells or tissues exhibit Her2 expression levels higher than normal baseline. Her2 overexpression can be at least 10% higher than the normal baseline level, or more preferably 20% higher, or more preferably 30% higher. As used herein, the term "Her2 overexpression" is refers to any level of Her2 expression that is greater than the normal baseline level. As indicated in the Background section, various cancers are characterized by overexpression of Her2. A Basal level of Her2 expression is typically that measured in tissues and non-cancerous cells expressing Her2.

The antibodies of the invention bind to a epitope of Her2, so that the binding of place to dimerization of Her2, phosphorylation of Her2 and cell apoptosis. As used here, the term "epitope" refers to a region of Her2 that binds to an antibody and that is protected from binding to a second antibody. In a preferred embodiment, the epitope is defined by the binding of mAb74 to Her2. This epitope is different from epitopes recognized by other anti-Her2 antibodies (see Table 1). It is remarkable that other antibodies anti-Her2 induce dimerization and phosphorylation of Her2, but not apoptosis, and that they recognize epitopes on Her2 that They are different from those recognized by mAb74.

The antibodies of the invention can be polyclonal or monoclonal or fragments thereof. The murine polyclonal and monoclonal antibodies are produced by standard immunological techniques. Antibody fragments encompass those antibodies that interact specifically with Her2 and induce apoptosis in cells and tissues that express Her2. As indicated in the examples below, there is a correlation between the apoptotic activity of mAb74 and the phosphorylation and dimerization of Her2 receptors. Thus, it is preferred that the antibody fragments of the invention retain their bivalent structure, which is likely to promote dimerization and activation of receptors. Also included antibodies produced by recombinant means, such as chimeric antibodies (variable region and constant region derived from different species) and antibodies grafted with "CDR" (complementarity determining region derived from a different species), as described in US Pat. No. 4,816,567 and 5,225,539. Preferably, the antibodies are at least partly of human origin. These include humanized antibodies, typically produced by recombinant methods, where Human sequences constitute all or part of the antibody. Too fully human antibodies produced in mice are included genetically altered (see PCT Application No. 93/12227).

The antibodies of the invention may have also a detectable label attached thereto. The marking can be a fluorescent, affinity or isotopic label. As examples fluorescein isothiocyanate (FITC) is included for detection of fluorescence, horseradish peroxidase, which allows the Excision detection of a chromogenic substrate, radioisotopes such as I125 for autoradiography detection and avidin / biotin for antibody detection and purification by affinity of antigens and antigen carrying cells.

Also included in the invention are lines. hybridoma cell producing a monoclonal antibody, where the antibody induces apoptosis in cells and tissues that express Her2. In one embodiment, hybridoma produces an antibody monoclonal that recognizes an epitope in Her2, so that a Her2-antibody complex results in induction of apoptosis Preferably, the hybridoma produces an antibody that recognizes the epitope on Her2 that is recognized by mAb74. The line hybridoma cell that produces mAb74 has been deposited in the American Type Culture Collection, Rockville, MD, April 4, 1996 under Accession No. ATCC No. HB-12078.

Various cancers are characterized by high levels of Her2 expression, including breast, ovarian, prostate, gastric and colorectal cancers (Press et al., In Effects of Therapy on Biology and Kinetics of The Residual Tumor , Part A: Preclinical Aspects, pp. 209-221 (1990); Fukushige et al., Mol. Cell. Biol. 6 , 955-958 (1986); Bargmann et al., In The Oncogene Handbook , pp. 107-119 (1988)) . A correlation between a poor prognosis and the overexpression of Her2 in cancerous tissue has been described. Patients with a poor prognosis typically have a higher recurrence and a higher incidence of mortality. Frequently, such patients may benefit from an aggressive treatment regimen that includes high doses of chemotherapy. Such therapy is expensive and may present risks for the patient. It has been proposed to use antiHer2 antibodies in a cancer treatment regimen to inhibit tumor growth where antibodies are used together with cytotoxic agents. An approach involves combinations of antiHer2 antibodies and chemotherapeutic agents (such as cisplatin, 5-fluorouracil and others) to increase the cytotoxic effect of chemotherapeutic drugs (this effect is referred to as antibody-dependent cellular cytotoxicity or "ADCC"). A second approach employs immunotoxins or antibody conjugates with cytotoxic agents, such as various A-chain toxins, ribosome inactivating proteins and ribonucleases. Another approach involves the use of biospecific antibodies designed to induce cell mechanisms for tumor death (see, for example, U.S. Patent Nos. 4,676,980 and 4,954,617).

The antibodies of the present invention are in themselves toxic to cells expressing induction Her2 of apoptosis They can be used advantageously in the treatment of cancer characterized by overexpression of Her2, such as cancer breast, ovarian, gastric, prostate and colorectal. The use of antibodies have significant advantages over previous approaches, in the sense that the administration of cytotoxic agents, which are deleterious to all growing cells. It is anticipated that the use of antibodies alone to treat cancer will greatly reduce the unwanted side effects associated with the administration of high doses of cytotoxic agents or combinations of chemotherapy / antibody combination therapy. Alternatively, if a cytotoxic agent is used, it is expected that the use of the present antibodies together with cytotoxic agents be advantageous in the sense that less agent can be used cytotoxic to achieve the same therapeutic effect. It can administer an antibody such as mAb74 alone or in combination with other anti-Her2 antibodies that induce apoptosis

The route of administration is expected for Antibodies of the invention are parenteral. The administration can be by subcutaneous, intravenous or intramuscular injection and may be an injection in a single bolus or by continuous infusion. The amount of antibody used will vary depending on the nature and the severity of the condition, but, in general, will vary between about 0.1 µg / kg body weight and about 100 mg / kg body weight.

The invention provides a pharmaceutical composition consisting of a therapeutically effective amount of an anti-Her2 antibody that induces apoptosis with a pharmaceutically acceptable adjuvant. The adjuvant is selected from one or more of a diluent, carrier, preservative, emulsifier, antioxidant and / or stabilizer. Pharmaceutically acceptable adjuvants are known to one skilled in the art and extensive review of them is done in Remington's Pharmaceutical Sciences , 18th ed., AR Gennaro, ed., Mack, Easton, PA (1990). The pharmaceutical compositions are sterile, non-pyrogenic and suitable for injection. As used herein, a "therapeutically effective amount" refers to that amount of antibody that provides a therapeutic effect for a given condition and administration regime. In the present invention, a therapeutic effect is the induction of apoptosis in tumors characterized by Her2 overexpression. Antibodies are preferably those that do not elicit an immune response when administered to a patient in need of treatment. In one embodiment, the antibodies are human or humanized antibodies that can be prepared using methods known to one skilled in the art.

The following examples are offered for further illustration of the invention, but are not intended to be limiting of its scope.

Example 1 Production of extracellular domains Her2, Her3 and Her4 Cloning and expression of the Her2 extracellular domain (Her2 soluble)

A Her2 receptor construct was prepared soluble as follows. A full length cDNA clone was digested Her2 in plasmid pLJ (pLJ is described in Korman et al., Proc. Natl Acad. Sci. USA 84, 2150-2054 (1987) with AstII, which cuts once at position 2107 of the DNA sequence Her2 (numbering according to Coussens et al., Cited above). He cut himself linearized plasmid with HindIII, which cuts 5 'of the ATG from initiation, to release a fragment of approximately 2200 bp. This fragment was cloned into pDSRα2 5'-HindIII to 3'-SalI using an oligonucleotide binder (AstII-SalI) that contained a FLAG sequence inside of frame and a codon of completion of the translation. CDNA resulting encodes for extracellular ligand binding domain Her2, which occupies amino acid residues 1-653, merged to the FLAG sequence (underlined):

Thr Ser Asp Tyr Lys Asp Asp Asp Asp Lys STOP

This construct was transfected into CHOd- cells. Single cell clones were derived from the selected population and were studied regarding the production of soluble Her2 by Western blot analysis anti-FLAG and anti-Her2.

Cloning and expression of the Her3 extracellular domain (Her3 soluble)

A cDNA clone containing the complete sequence of Her3 studying a prepared cDNA library from SKBR3 (American Type Tissue Collection, Bethesda, MD, ATCC HTB 30). The library was divided into 49 pools containing each 3,200 individual clones. Plasmid DNA was transferred from each pool to a nitrocellulose filter (Schleicher and Schuell, Keene, NH). Two oligonucleotide probes were synthesized corresponding to the 3 'end of the Her3 sequences:

5'-CCACCCGGGTTAGAGGAAGA-3 ' Y

5'-AGTTACGTTCTCTGGGCATTA-3 '

and they were used to study the filters of the SKBR3 cDNA library. Hybridization was performed in SSC 6X, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, SDS 0.2%, 2 mM EDTA, 2X Denhardt solution and 50 mg / ml DNA of Salmon sperm at 42 ° C for 16 hours. The washings were then washed 42 ° C filters with 2X SSC, 0.2% SDS, 2 mM EDTA for 30 minutes and were exposed to X-ray films at -80 ° C for 2 days.

Ten pools were also characterized that gave positive signals in hybridization by reaction analysis in polymerase chain ("PCR") to determine if also encoded for the 5 'sequence of Her3. DNA was amplified plasmid of each pool with oligonucleotide primers corresponding to the 5 'end of the Her3 sequences:

5'-CATGAGGGCGAACGACGCTCTG-3 ' Y

5'-CTTGGTCAATGTCTGGCAGTC-3 '

PCR was carried out for 40 cycles, each cycle at 94 ° C, 30 seconds; 50 ° C, 30 seconds, and 72 ° C, 30 seconds. Three of the ten poles contained a full-length Her3 cDNA. The three pools were re-studied by the procedure of colony hybridization of Lin et al. (Gene 44, 201-209 (1986)) until obtaining unique clones of each pool. CDNA sequencing revealed an identical sequence to the published (Kraus et al., cited above).

Plasmid pJT2-Her3 was used to PCR amplification of the soluble Her3 domain using the following primers:

Sense: 5'-CGCTCTAGACCACCATGAGGGCGAACGACGCTCTGCA-3 ' Antisense: 5'-CGCGGATCCGTCGACTCACTATGTCAGATGGGTTTTGCCGAT-3 '

After digestion with the enzymes of XbaI and SalI restriction, the 1.9 kb PCR fragment was subcloned in pDSRα2 (PCT Patent Application No. WO91 / 05795), which He had been split with XbaI and SalI. The sequences were confirmed Her3 in the resulting plasmid by DNA sequencing. The plasmid pDSRα2 / Her3 to transfect CHOd - cells for the expression of soluble Her3.

Cloning and expression of the Her4 extracellular domain (Her4 soluble)

A Her4 cDNA clone in length was obtained completed by study of a human fetal brain cDNA library (Stratagene, San Diego, CA). Two Her4 cDNA probes were prepared by PCR amplification of human brain cDNA (Clontech Laboratories, Inc., Palo Alto, CA). The cDNA probe 1 corresponds to the sequences of the 5 'end of Her4 encoding the residues  of amino acid 32 to 177 and the cDNA probe 2 corresponds to the 3 'end sequences of Her4 encoding the residues of amino acid 1137 to 1254. (Plowman et al., cited above). I know studied approximately 4 X 10 6 pfu from the cDNA library of human fetal brain sequentially with the 5 'end probe of Her4 and the probe of the 3 'end of Her4. The solution of Hybridization contained 6X SSC, 50 mM sodium phosphate (pH 6.8), SDS 0.2%, 2 mM EDTA, 0.1% sodium pyrophosphate, solution of Denhardt 2X, 50 mg / ml salmon sperm DNA and formamide at fifty%. Hybridization was done at 42 ° C for 16 hours. They washed filters at 67 ° C with 2X SSC, 0.2% SDS, 2 mM EDTA for 60 minutes and then they were exposed to X-ray films at -80 ° C during night. Autoradiography of the filters showed that 12 clones were hybridized with the 5 'end probe and another 5 clones were they hybridized with the 3 'end probe. The clones were purified simple by new plating, they were studied by hybridizations of probes as described and the clones were sequenced positive.

All sequenced positive cDNA clones They turned out to be partial Her4 cDNA clones. The sequences turned out to be identical to the published Her4 sequence (Plowman et al., cited above), except for a short deletion / substitution in the extracellular domain. Amino acids 626 to 648 of the Published sequence of Her3 (NGPTSHDCIYYPWTGHSTLPQHA) were substituted by the IGSSIEDCIGLMD peptide sequence. In addition, G in amino acid position 573 of the Plowman sequence remained replaced by D.

Since none of the 17 clones contained the Her4 full-length cDNA, two overlapping clones were fused together to generate a full-length Her4 receptor using the techniques described by Maniatis et al. ( Molecular Cloning: A Laboratory Manual , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory (1982)). One clone encoded for amino acid residues of Her4 1 to 738 and another encoded for amino acid residues 588 to 1298. These two overlapping clones were released from plasmid pBluescriptSK by digestions with restriction enzymes and mounted on plasmid pGEM4 to generate a cDNA. Her4 full length.

A soluble Her4 receptor was constructed by PCR amplification of a 700 bp coding Her4 fragment of amino acids 409 to 639 of the full-length cDNA of Her4. The sequences of the two primers used in this amplification they were:

5'-CCAAACATGACTGACTTCAGTG-3 ' Y

5'-GGCCAATTGCGGCCGCTTACTAATCCATCAGGCCGATGCAGTCTTC-3 ' Y

PCR was carried out for 25 cycles, being each cycle at 94 ° C, 30 seconds; 55 ° C, 30 seconds, and 72 ° C, 30 seconds. This 700 bp PCR product was purified by agarose gel electrophoresis. Plasmid pGEM4 / Her4 was digested with Not I and BstE II to produce two fragments: one of them contained plasmid pGEM4 and the cDNA of the 5 'end of Her4 encoder of the extracellular domain of the receptor from the amino acid 1 to 420 and the second fragment extended from amino acid 421 of Her4 to the end of the Her4 molecule. These two DNA fragments were separated on agarose gel and were recovered the 5 'end fragment of pGEM4 / HER4. He digested the 700 bp PCR fragment Her4 with BstE II and Not I and ligated with the 5 'end fragment of pGEM4 / HER4. The resulting cDNA encodes for the extracellular domain of the Her4 receptor, which is extends along amino acid residues 1 to 639. It sequenced the portion amplified by PCR to confirm that it was not they had produced errors in the PCR

Her4 cDNA construction was released soluble from plasmid pGEM4, was inserted into plasmid pDSRa2 and was transfected into CHOd - cells using standard techniques (Maniatis et al., cited above). Clones from a single cell were derived from the population selected and studied in terms of the production of Her4 soluble by BIAcore analysis.

Purification of the sHer2, sHer3 and sHer4 receptors

CHO cell conditioned medium was concentrated expressing soluble Her2 (sHer2) 12.5 times with a device Pellicon tangential flow ultrafiltration (Amicon) equipped with a 50 K MWCO filter cassette (Filtron Technology) and diafiltered the concentrate with three volumes of 20 mM potassium phosphate, NaCl 100 mM, pH 6.8. The diafiltered concentrate was mixed with Hydroxyapatite (Calbiochem) balanced in diafiltration buffer. The unbound fraction was diluted with equal volume of water and then applied to a flow Q-Sepharose column Fast (Pharmacia) balanced in 10 mM potassium phosphate, 50 NaCl mM, pH 7.0. The column was eluted with a linear gradient of NaCl 50-600 mM. A pool was made with the fractions that They contained> 95% of sHer2. SHer3 and sHer4 were also purified from conditioned media of CHO cells expressing these proteins in a manner similar to the procedure described above. Because of his higher value of pI, sHer3 joined, and eluted from, a column of Q-Sepharose balanced in potassium phosphate 10 mM, 50 mM NaCl, pH 7.5.

Example 2 Production of anti-HER2 antibodies

Procedures for immunizing animals, preparing fusions and making a selective study of hybridomas and purified antibodies were carried out as generally described in Harlow and Lane, Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory (1988).

Enzyme-linked immunosorbent assay ("EIA")

96-well plates were coated with 2 \ mug / ml of sHer2, 2 \ mug / ml of sHer3 or 2 \ mug / ml of sHer4 in a carbonate-bicarbonate buffer. After block, hybridoma conditioned medium was added to the plate and incubated for 2 hours. The medium was aspirated and the plates washed before the addition of rabbit IgG antibody anti-mouse conjugate with horseradish peroxidase Spicy (Boehringer Mannheim). After one hour of incubation, the plates were aspirated and washed five times. It was detected antibody bound with ABTS color reagent (Kirkegaard and Perry Labs., Inc.). The degree of antibody binding was determined monitoring the absorbance increase at 405 nm.

Cloning and determination of the IgG subtype

A single cell cloning was done in a 96-well plate using a limiting dilution method. I know studied the conditioned media of single cell clones in as for the production of antibodies using the EIA described above. The major antibody-producing clones were chosen for the cell growth expansion studies, determination of Subtype and competition.

BIAcore analysis

SHer2, sHer3 or sHer4 were covalently coupled purified to a CM5 sensor chip through the primary amine group using 40 µl of the receptor in 10 mM Na acetate, pH 4.0 (10 receiver mug per ml). Groups not blocked reacted on the sensor chip with an injection of 50 µl of 1 M ethanolamine hydrochloride (Pharmacia Biosensor AB). Every cycle of analysis consisted of an injection of 40 µl of supernatant of hybridomas (or purified mAbs), followed by injection of 10 µL of 10 mM HCl, to regenerate the chip. The binding of was detected mAbs for the change in SPR, measured in resonance units ("RU"). For most proteins, 1,000 RU correspond at a surface concentration of about 1 ng / mm2.

Preparation and study of hybridoma cell lines

7 Balb / c mice were injected subcutaneously three times at three week intervals with 10 \ mug of Her2 soluble. The protein was emulsified with RIBI adjuvant. They were evaluated serum titers for Her2 at 8 weeks and were selected the two mice with the highest titles and were given a final IV injection of 10 µg of soluble Her2. Three days later, the two mice were sacrificed and the spleens were removed, they broke into a Stomacher tissue blaster and filtered and simple cells were recovered. After three washes, it counted spleen cells, mixed with myeloma cells mouse (SP2 / 0) in a 3: 1 ratio (spleen: SP2 / 0) and merged in the presence of 50% PEG (MW-molecular weight- 1,500). The fused cells were plated on a total of 10 plates of 96 wells at a concentration of spleen cells of 1.25 X 10 5 per well in a medium consisting of DMEM: RPMI (1: 1), FBS at 10% and ORIGIN at 10%. The selection of fused cells was carried out in the middle of HAT selection. The media were studied of culture by EIA regarding antibodies to Her2 after viable cell colonies will occupy approximately 30% of the well. 68 positives were identified in 960 wells. They cloned 43-well cells by limiting dilution to produce single cell colonies. Wells containing were marked unique colonies and, when they grew to 30% of the well area, they studied regarding anti-Her2 antibodies by EIA and BIAcore. The final number of clones of a single cell was 26, which represents 20 original standard wells.

Based on the union of the supernatants of hybridomas to sHer2 studied by EIA and BIAcore, 10 were selected clones for further study. 5 X 10 6 cells were injected of each of the 10 clones in primed Balb / c mice and collected ascites fluid at approximately 10 days. I know purified affinity immunoglobulins on a column of MAPS II protein (BioRad). IgG antibodies were studied purified by EIA regarding the union to Her2, Her3 and Her4 as It has been described above. Binding capacity was evaluated at 10 ng / ml or 100 µg / ml of mAbs. The binding of antibodies to sHer2 was readily apparent at an antibody concentration of 10 ng / ml, while the union to sHer3 and sHer4 was insignificant even to an antibody concentration of 100 µg / ml. The data show that all clones, except mab83, bind strongly to sHer2 without detectable binding to sHer3 and sHer4.

IgG subtypes were determined in hybridoma supernatants using a Ab-Stat-Isotype Kit (Sangstate Medical Corp.) and Table I show the results.

Binding of mAbs to sHer2, sHer3 and sHer4

The binding of mAbs to sHer2 on a chip was investigated BIAcore using 10 µg / ml of mAbs and was evaluated as units of resonance (RU). As shown in Table I, two clones (52 and  58) showed more than 1,000 RU, 2 clones (35 and 42B) showed around 700 RU, 2 clones (43A and 74) showed around 300 RU, 2 clones (83 and 97) showed around 100 RU and 2 clones (29 and 86) showed less than 100 RU. The results indicated a broad affinity range among the ten clones. No union was observed detectable from anti-sHer2 to sHer3 and sHer4 mAbs. These Results, together with the EIA data, confirm that mAbs generated against sHer2 specifically bind sHER2 with little or no union to sHer3 and sHer4.

(Table goes to page next)

one

Competitive epitope assay

The epitopic specificity of the anti-sHer2 mAbs binding antibody pairs monoclonal simultaneously to sHer2 immobilized on a chip BIAcore MAbs directed against different epitopes must bind independently of each other, while directed mAbs against closely related epitopes should interfere sterically in the union of each other. The first mAb was injected three times in a volume of 40 µl at a concentration 10 µg / ml on the surface of immobilized sHer2. I know they then injected 40 µl of the second mAb and the ability to simultaneously join sHer2. It regenerated the Biosensor surface by injection of 10 µL of 50 mM HCl. Binding was also analyzed when the sequence was reversed. injection of each pair of mAbs. This analysis divided the mAbs into 4 different groups of epitopic specificity, as shown in Table I. There was no apparent correlation between the epitope clustering and phosphorylation activity, except for mAb74, which seems to have a unique epitope with respect to the others mAbs

Example 3 Characterization of mAb74 epitope in Her2

The effect of glycosylation on the the interaction of mAb74 with sHer2 as follows. 60 were denatured mug of sHer2 in 20 mM BTP, 40 mM NaCl, pH 7.4, for five minutes in a boiling water bath in the presence of 0.4% SDS. After denaturation, NP-40 was added (Boehringer Mannheim) at 2% v / v and the reaction was diluted with equal volume of H 2 O DI before adding 3 units of Recombinant N-glycanase (Genzyme). He let the reaction proceed with gentle stirring at 37 ° C for 20 h.

A detection system kit of ECL glycoproteins (Amersham Life Science) to determine the degree of deglycosylation. 0.25 µg of each of sHer2 and of sHer2 deglycosylated to a 4-20% gel (Novex) under conditions not reducing and then deposited in nitrocellulose (Schleicher & Schuell) for 1 hour at 90 volts in one device Bio-Rad mini PROTEAN II (BioRad) with cooling. After depositing, the membrane was treated with metaperiodate of 10 mM sodium for 20 minutes and then 300 nM biotin hydrazide for 60 minutes, both in 100 mM sodium acetate, pH 5.5, at room temperature. After each stage, the membrane was washed with Three changes of PBS. Dried non-fat milk (Carnation) was added to PBS at a concentration of 5% (w / v) and incubated overnight at 4 ° C to block nonspecific binding. The membrane was incubated at room temperature with radish streptavidin peroxidase spicy conjugated with ECL detection reagents for one minute. The spot was exposed to Hyperfilm-ECL (Amersham Life Science) No protein band was observed in the sample deglycosylated (Figure 1A), indicating that it had occurred A complete deglycosylation.

SHer2 intact and deglycosylated (25 ng) of each) and were taken to a 4-20% gel (Novex) in reducing and non-reducing conditions. The gel was dried for 1 h at 90 volts, it was blocked with dried 5% nonfat dry milk and detected with 0.4 µg / ml of mAb74, followed by 1/5000 of horseradish peroxidase conjugated anti-mouse after three 10 minute washes in PBS, 0.1% Tween 20. I know used an ECL kit (Amersham Life Science) for detection. I know observed that mAb74 bound both glycosylated sHer2 and deglycosylated under non-reducing conditions (Figure 1B). I dont know observed antibody binding under reducing conditions.

Example 4 Dimerization of Her2 by antibodies anti-Her2

Typically, antibodies have two sites of binding for antigens, so that antibodies can be expected that bind to receptors can promote dimerization of receivers Size exclusion chromatography was used ("SEC") with light scattering detection to determine stoichiometry of antibody binding anti-Her2 to sHer2. The use of SEC with dispersion of online light has advantages over the SEC alone to determine the molecular weight or stoichiometry of a protein complex. While the elution position of a protein or complex is indicative of molecular weight using conventional SEC, a measurement of light scattering is independent of the position of elution of a protein or complex. In addition, the molecular weight obtained from light scattering reflects only the polypeptide if the extinction coefficient of the polypeptide is used only in the analysis. The online light scattering system / chromatography size exclusion uses three detectors in series: a light scattering detector (Wyatt Minidawn), a detector of the refractive index (Polymer Laboratories PL-RI) and a UV absorbance monitor at 280 nm (Knauer A293). A column of SEC Superdex 200 (Pharmacia) was used balanced with phosphate buffered saline ("PBS") of Dulbecco and a sample handle of 100 µl. The system was operated on a flow rate of 0.5 ml / min. MAb complexes anti-sHer2 and sHer2 were prepared by mixing 55 mul 1.5 mg / ml mAb35, 0.8 mg / ml mAb52, 1.2 mg / ml mAb58, 1.6 mg / ml of mAb42, 0.84 mg / ml of mAb74 and 0.89 mg / ml of mAb83 with 55 µL of 2.0, 2.0, 1.3, 2.0, 2.0 and 2.0 mg / ml of sHer2, respectively. The complexes of the previous mAbs and sHer3 were prepared in a similar way. 100 µL samples were injected of each complex in a Superdex 200 column and the elution using light scattering detectors, index of refraction and UV absorbance.

For a glycoprotein complex, the molecular weight of its polypeptide is proportional to (uv) (LS) / [e_ {p (RI) 2] (Takagi, J. Chromatogr. 506 , 409-446 (1990); Arakawa et al., Arch. Biochem. Biophys. 308 , 267-273 (1994); Philo et al., J. Biol. Chem. 269 , 27840-27846 (1994)), where uv, LS and RI are the signals of the absorbance, light scattering and refractive index detectors, respectively and e_ {p} is the extinction coefficient (the absorbance of a 1 mg / ml solution for a 1 cm path) of the polypeptide. For a complex with a known stoichiometry (A_ {m} B_ {n}), its extinction coefficient can be calculated with the equation E_ {p} = (mxE_ {A} xM_ {A} + nxE_ {B} M_ {B }) / (mxM_ {A} + nxM_ {B}), where E_ {A}, E_ {B}, M_ {A} and M_ {B} are the extinction coefficient of the polypeptide and the molecular weight of protein A or from B.

In order to obtain the molecular weight and the stoichiometry of a glycoprotein complex, its extinction coefficient However, the extinction coefficient of a complex cannot be calculated unless the stoichiometry A consistent method is used to solve this problem, assuming several possibilities for stoichiometry of the complex. For each known stoichiometry, a extinction coefficient and the corresponding molecular weight experimental. Finally, stoichiometry is selected with the better consistency between experimental molecular weight and theoretical as correct stoichiometry for the complex. In the table II the results of this method are given.

TABLE II Binding of mAb to sHer2 determined by SEC / dispersion of the light

two

3

 * The molecular weights (MW) of the table reflect only the polypeptide.

Experimental molecular weights (with carbohydrate exclusion) for complexes are more consistent with the theoretical values assuming 2 sHer2 for 1 mAb for each of the 5 mAbs studied. This shows that these antibodies could dimerize Her2 expressed on the surface mobile. However, as sHer2 and mAbs were mixed at 2: 1, The observed results do not exclude the possibility of training of a complex of 1 sHer2: 1 mAb when the mAb is present in excess. No complex was observed for the mixture of sHer2 and mAb83. The cause of this may be weak union and dissociation of the complex during the chromatographic procedure. The samples containing sHer2: mAb in a 2: 1 molar ratio eluted as a single peak, suggesting the formation of a complex of 2 sHer2: 1 mAb without dissociation during elution.

To verify that these antibodies do not dimerize on Her3, similar experiments were performed using mixtures of mAbs and sHer3. No complexes were detected between sHer3 and any of the mAbs

Example 5 Phosphorylation of antibody receptors anti-Her2

Adherent cells were grown (SKBR3 or MDAMB453) in 48-well plates and washed with DMEM 2-3 times A pellet of the cells was obtained in suspension (32D, Her2 / 32D, HEG / 32D) by centrifugation and washed with PBS. HEG / 32D is a cell line transfected with a receptor chimeric Her2 / EGF (HEG) that has an extracellular domain of Her2 covering amino acid residues 1-653 and intracellular and transmembrane domains of the EGF receptor that encompass amino acid residues 646-1210. I know added mAb or control ligand solution to the well or tube the pellet and incubated for 5 minutes at 37 ° C. The solution and the cells were solubilized with SDS sample buffer. Samples were subjected to SDS-PAGE, followed by Western blotting and probing with anti-phosphotyrosine

Twelve clones of mAbs were studied anti-sHer2 in terms of stimulation of phosphorylation of receptor tyrosine in SKBR3 cells. Such as shown in Figure 2-a, mAb74, 52 and 83 potently stimulated tyrosine phosphorylation of 180-185 kDa proteins in SKBR3 cells where both Her2 and Her3 were identified. Phosphorylation was dose dependent (Figure 2-b). As it shown in Figure 3, phosphorylation of SKBR3 cells by mAb 74, 52 and 83 was inhibited with sHer2. To determine what receptor phosphorylates, Her2 and Her3 were immunoprecipitated from SKBR3 and Her2, Her3 and Her4 were immunoprecipitated from MDAMB453 after incubating the mAbs and analyzed by Western blots probed with anti-phosphotyrosine. Her2 and Her3 in SKBR3 or Her2, Her3 and Her4 in MDAMB453 were all phosphorylated in tyrosine

A similar test has been conducted with lines transfected cells, Her2 / CHO and Her2 / 32D, to study the direct interaction of mAb and Her2. MAbs 52, 74 and 83 do not they managed to stimulate phosphorylation of Her2 in cells Transfected Her2 / CHO and Her2 / 32D (Figure 4 shows the data for Her2 / 32D cells only). On the contrary, the receiver Chimeric Her2 / EGF was phosphorylated in HEG / 32D (Figure 4). It has been made a subsequent experiment using a Her2 / 32D transfectant that expressed Her2 at levels comparable to those of the chimeric receptor HEG shown in Figure 4. Under these conditions, mAb74 stimulates phosphorylation of Her2 in Her2 / 32D cells. The results suggest that mAb74 activates Her2 kinase by homodimerization in Her2 / 32D cells, but which can be activated by heterodimerization in SKBR3 cells.

Example 6 Morphological change and antibody-induced cell apoptosis anti-Her2 Cell morphological change

Cells were seeded in 5 cm plates until one confluence of approximately 20% and mAbs were added after from 18 h. After 5 days, the cells were observed with Optical microscopy, photographed and counted.

Her2 / MCF7 cells were incubated with 250 nM of mAb42b, mAb83 and mAb74. After 5 days of incubation, mAb74 caused extensive cell death and a dramatic change in morphology cellular, primarily cell elongation, as shown in Figure 5. mAb83 caused a moderate change in cell morphology and 42b resulted in little change. The number of viable cells after of incubation with mAb74 with Her2 / MCF7 cells for five days it was only 36% of the control not subjected to incubation with mAb. mAb74 also induced cell morphological changes in MDAMB43 cells (Figure 5F).

Cell apoptosis

Cells were seeded in an inclined 8-well chamber (Nunc) to a confluence of approximately 60-70% and, 18 h later, the culture medium was changed to medium containing 1% FBS with or without mAb. On day 1, the cells were fixed with 4% neutral buffered formalin ("NBF"), followed by three washes with PBS. After drying the cells, apoptosis was detected using a modified TUNEL method. TUNEL detects the 3'-OH DNA ends generated by DNA fragmentation by marking the ends with dUTP conjugated with digoxigenin using terminal deoxynucleotidyl transfer and then incubating with horseradish peroxidase-conjugated anti-digoxigenin ("HRP"). HRP bound to the substrate, 3-amino-9-ethylcarbazole (Sigma) was detected. Most of the reagents used came from the Apop Tag in situ apoptosis detection kit (Oncor). The antibodies conjugated to HRP were from Boehringer Mannheim.

We saw that mAb74 has the most powerful effect on phosphorylation of receptor tyrosine (Fig. 1A), change in cell morphology (Fig. 5) and cell death. To clarify the mechanism of cell death caused by mAb74, we examine the apoptosis by a modified TUNEL method. As shown in the Figure 6, cells incubated with mAb74 for one day showed apoptosis, as detected by the red color using the method TUNEN, while incubation with mAb42b was sparsely apoptotic in MDAMB453 and Her2 / MCF7 (MCF7 cells transfected with Her2 full length). The number of apoptotic cells Induced by 50 mM mAb74 was approximately 10% of the number 500 nM mAb74 induced, indicating that mAb74 apoptosis It is dose dependent (Figure 6). mAb74 also induced apoptosis in Her2 / MCF7 cells. After 5 days of incubation with mAb74, the live cells were still present in culture, but could not detect apoptosis, suggesting that apoptotic cells are they had detached and living cells were not suffering a apoptotic process The surviving cells had experienced morphological changes, such as those seen in Figure 5.

(1. GENERAL INFORMATION:

         \ vskip0.666000 \ baselineskip
      

(i)

APPLICANT: AMGEN INC.

         \ vskip0.666000 \ baselineskip
      

(ii)

TITLE OF THE INVENTION: Antibody induced apoptosis

         \ vskip0.666000 \ baselineskip
      

(iii)

NUMBER OF SEQUENCES: 9

         \ vskip0.666000 \ baselineskip
      

(iv)

ADDRESS FOR THE CORRESPONDENCE:

         \ vskip0.333000 \ baselineskip
      

(TO)

ADDRESSEE: Amgen Inc.

         \ vskip0.333000 \ baselineskip
      

(B)

STREET: 1840 Dehavilland Drive

         \ vskip0.333000 \ baselineskip
      

(C)

CITY: Thousand Oaks

         \ vskip0.333000 \ baselineskip
      

(D)

STATE: California

         \ vskip0.333000 \ baselineskip
      

(AND)

COUNTRY: USA

         \ vskip0.333000 \ baselineskip
      

(F)

ZIP: 91230-1789

         \ vskip0.666000 \ baselineskip
      

(v)

READABLE FORM OF COMPUTER:

         \ vskip0.333000 \ baselineskip
      

(TO)

MEDIA TYPE: Floating disc

         \ vskip0.333000 \ baselineskip
      

(B)

COMPUTER: IBM PC compatible

         \ vskip0.333000 \ baselineskip
      

(C)

OS: PC-DOS / MS-DOS

         \ vskip0.333000 \ baselineskip
      

(D)

PROGRAM: PatentIn Release # 1.0, Version # 1.30

         \ vskip0.666000 \ baselineskip
      

(saw)

DATA OF THE CURRENT APPLICATION

         \ vskip0.333000 \ baselineskip
      

(TO)

NUMBER OF REQUEST:

         \ vskip0.333000 \ baselineskip
      

(B)

DATE REQUEST:

         \ vskip0.333000 \ baselineskip
      

(C)

CLASSIFICATION:

         \ vskip0.666000 \ baselineskip
      

(viii)

INFORMATION ABOUT THE LAWYER / AGENT:

         \ vskip0.333000 \ baselineskip
      

(TO)

NAME: Winter Ph.D., Robert B.

         \ vskip0.333000 \ baselineskip
      

(B)

REFERENCE / NUMBER OF RECORD: A-377

         \ vskip0.666000 \ baselineskip
      

(2) INFORMATION FOR SEQ ID NO: 1:

         \ vskip0.666000 \ baselineskip
      

(i)

CHARACTERISTICS OF SEQUENCE:

         \ vskip0.333000 \ baselineskip
      

(TO)

LENGTH: 10 amino acids

         \ vskip0.333000 \ baselineskip
      

(B)

KIND: amino acid

         \ vskip0.333000 \ baselineskip
      

(C)

TYPE OF HEBRA: simple

         \ vskip0.333000 \ baselineskip
      

(D)

TOPOLOGY: linear

         \ vskip0.666000 \ baselineskip
      

(ii)

TYPE OF MOLECULE: protein

         \ vskip0.666000 \ baselineskip
      

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:

         \ vskip1.000000 \ baselineskip
      

\ sa {Thr Ser Asp Tyr Lys Asp Asp Asp Asp Lys}

         \ vskip0.666000 \ baselineskip
      

(2) INFORMATION FOR SEQ ID NO: 2:

         \ vskip0.666000 \ baselineskip
      

(i)

CHARACTERISTICS OF SEQUENCE:

         \ vskip0.333000 \ baselineskip
      

(TO)

LENGTH: 20 pairs of bases

         \ vskip0.333000 \ baselineskip
      

(B)

TYPE: acid nucleic

         \ vskip0.333000 \ baselineskip
      

(C)

TYPE OF HEBRA: simple

         \ vskip0.333000 \ baselineskip
      

(D)

TOPOLOGY: linear

         \ vskip0.666000 \ baselineskip
      

(ii)

TYPE OF MOLECULE: CDNA

         \ vskip0.666000 \ baselineskip
      

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

\hskip-.1em\dddseqskip

CCACCCGGGT TAGAGGAAGA

\hfill

20

 \ hskip-.1em \ dddseqskip 

CCACCCGGGT TAGAGGAAGA

 \ hfill 

twenty

         \ vskip0.666000 \ baselineskip
      

(2) INFORMATION FOR SEQ ID NO: 3:

         \ vskip0.666000 \ baselineskip
      

(i)

CHARACTERISTICS OF SEQUENCE:

         \ vskip0.333000 \ baselineskip
      

(TO)

LENGTH: 21 pairs of bases

         \ vskip0.333000 \ baselineskip
      

(B)

TYPE: acid nucleic

         \ vskip0.333000 \ baselineskip
      

(C)

TYPE OF HEBRA: simple

         \ vskip0.333000 \ baselineskip
      

(D)

TOPOLOGY: linear

         \ vskip0.666000 \ baselineskip
      

(ii)

TYPE OF MOLECULE: CDNA

         \ vskip0.666000 \ baselineskip
      

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

\hskip-.1em\dddseqskip

AGTTACGTTC TCTGGGCATT A

\hfill

21

 \ hskip-.1em \ dddseqskip 

AGTTACGTTC TCTGGGCATT A

 \ hfill 

twenty-one

         \ vskip0.666000 \ baselineskip
      

(2) INFORMATION FOR SEQ ID NO: 4:

         \ vskip0.666000 \ baselineskip
      

(i)

CHARACTERISTICS OF SEQUENCE:

         \ vskip0.333000 \ baselineskip
      

(TO)

LENGTH: 22 pairs of bases

         \ vskip0.333000 \ baselineskip
      

(B)

TYPE: acid nucleic

         \ vskip0.333000 \ baselineskip
      

(C)

TYPE OF HEBRA: simple

         \ vskip0.333000 \ baselineskip
      

(D)

TOPOLOGY: linear

         \ vskip0.666000 \ baselineskip
      

(ii)

TYPE OF MOLECULE: CDNA

         \ vskip0.666000 \ baselineskip
      

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

\hskip-.1em\dddseqskip

CATGAGGGCG AACGACGCTC TG

\hfill

22

 \ hskip-.1em \ dddseqskip 

CATGAGGGCG AACGACGCTC TG

 \ hfill 

22

         \ vskip0.666000 \ baselineskip
      

(2) INFORMATION FOR SEQ ID NO: 5:

         \ vskip0.666000 \ baselineskip
      

(i)

CHARACTERISTICS OF SEQUENCE:

         \ vskip0.333000 \ baselineskip
      

(TO)

LENGTH: 21 pairs of bases

         \ vskip0.333000 \ baselineskip
      

(B)

TYPE: acid nucleic

         \ vskip0.333000 \ baselineskip
      

(C)

TYPE OF HEBRA: simple

         \ vskip0.333000 \ baselineskip
      

(D)

TOPOLOGY: linear

         \ vskip0.666000 \ baselineskip
      

(ii)

TYPE OF MOLECULE: CDNA

         \ vskip0.666000 \ baselineskip
      

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

\hskip-.1em\dddseqskip

CTTGGTCAAT GTCTGGCAGT C

\hfill

21

 \ hskip-.1em \ dddseqskip 

CTTGGTCAAT GTCTGGCAGT C

 \ hfill 

twenty-one

         \ vskip0.666000 \ baselineskip
      

(2) INFORMATION FOR SEQ ID NO: 6:

         \ vskip0.666000 \ baselineskip
      

(i)

CHARACTERISTICS OF SEQUENCE:

         \ vskip0.333000 \ baselineskip
      

(TO)

LENGTH: 37 pairs of bases

         \ vskip0.333000 \ baselineskip
      

(B)

TYPE: acid nucleic

         \ vskip0.333000 \ baselineskip
      

(C)

TYPE OF HEBRA: simple

         \ vskip0.333000 \ baselineskip
      

(D)

TOPOLOGY: linear

         \ vskip0.666000 \ baselineskip
      

(ii)

TYPE OF MOLECULE: CDNA

         \ vskip0.666000 \ baselineskip
      

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

\hskip-.1em\dddseqskip

CGCTCTAGAC CACCATGAGG GCGAACGACG CTCTGCA

\hfill

37

 \ hskip-.1em \ dddseqskip 

CGCTCTAGAC CACCATGAGG GCGAACGACG CTCTGCA

 \ hfill 

37

         \ vskip0.666000 \ baselineskip
      

(2) INFORMATION FOR SEQ ID NO: 7:

         \ vskip0.666000 \ baselineskip
      

(i)

CHARACTERISTICS OF SEQUENCE:

         \ vskip0.333000 \ baselineskip
      

(TO)

LENGTH: 42 pairs of bases

         \ vskip0.333000 \ baselineskip
      

(B)

TYPE: acid nucleic

         \ vskip0.333000 \ baselineskip
      

(C)

TYPE OF HEBRA: simple

         \ vskip0.333000 \ baselineskip
      

(D)

TOPOLOGY: linear

         \ vskip0.666000 \ baselineskip
      

(ii)

TYPE OF MOLECULE: CDNA

         \ vskip0.666000 \ baselineskip
      

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

\hskip-.1em\dddseqskip

CGCGGATCCG TCGACTCACT ATGTCAGATG GGTTTTGCCG AT

\hfill

42

 \ hskip-.1em \ dddseqskip 

CGCGGATCCG TCGACTCACT ATGTCAGATG GGTTTTGCCG AT

 \ hfill 

42

         \ vskip0.666000 \ baselineskip
      

(2) INFORMATION FOR SEQ ID NO: 8:

         \ vskip0.666000 \ baselineskip
      

(i)

CHARACTERISTICS OF SEQUENCE:

         \ vskip0.333000 \ baselineskip
      

(TO)

LENGTH: 22 pairs of bases

         \ vskip0.333000 \ baselineskip
      

(B)

TYPE: acid nucleic

         \ vskip0.333000 \ baselineskip
      

(C)

TYPE OF HEBRA: simple

         \ vskip0.333000 \ baselineskip
      

(D)

TOPOLOGY: linear

         \ vskip0.666000 \ baselineskip
      

(ii)

TYPE OF MOLECULE: CDNA

         \ vskip0.666000 \ baselineskip
      

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

\hskip-.1em\dddseqskip

CCAAACATGA CTGACTTCAG TG

\hfill

22

 \ hskip-.1em \ dddseqskip 

CCAAACATGA CTGACTTCAG TG

 \ hfill 

22

         \ vskip0.666000 \ baselineskip
      

(2) INFORMATION FOR SEQ ID NO: 9:

         \ vskip0.666000 \ baselineskip
      

(i)

CHARACTERISTICS OF SEQUENCE:

         \ vskip0.333000 \ baselineskip
      

(TO)

LENGTH: 46 pairs of bases

         \ vskip0.333000 \ baselineskip
      

(B)

TYPE: acid nucleic

         \ vskip0.333000 \ baselineskip
      

(C)

TYPE OF HEBRA: simple

         \ vskip0.333000 \ baselineskip
      

(D)

TOPOLOGY: linear

         \ vskip0.666000 \ baselineskip
      

(ii)

TYPE OF MOLECULE: CDNA

         \ vskip0.666000 \ baselineskip
      

(xi)

DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9:

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

\hskip-.1em\dddseqskip

GGCCAATTGC GGCCGCTTAC TAATCCATCA GGCCGATGCA GTCTTC

\hfill

46

 \ hskip-.1em \ dddseqskip 

GGCCAATTGC GGCCGCTTAC TAATCCATCA GGCCGATGCA GTCTTC

 \ hfill 

46

Claims (19)

1. An anti-Her2 antibody or fragment thereof that induces apoptosis in cells that express Her2 2. The antibody of Claim 1, which recognizes an epitope on a Her2 polypeptide that is recognized by the monoclonal antibody produced by the cell line of ATCC hybridoma No. HB-12078. 3. The antibody of Claim 1, which is a monoclonal antibody. 4. The antibody of Claim 1, which is a humanized antibody. 5. The antibody of Claim 1, which is a human antibody 6. A hybridoma cell line capable of produce the antibody of Claim 3. 7. The antibody of Claim 1, wherein the fragment is an F (ab) or Fab 'fragment. 8. An antibody produced by the cell line of ATCC hybridoma No. HB-12078. 9. ATCC hybridoma cell line No. HB-12078. 10. The antibody of Claim 1, wherein The cells that express Her2 are tumor cells. 11. The antibody of Claim 10, wherein tumor cells derive from cancers of the lung, ovary, prostate, gastric and colorectal. 12. An in vitro method for inducing apoptosis in cells expressing Her2, consisting of administering an amount of the antibody of claim 1 sufficient to induce apoptosis. 13. The in vitro method of Claim 12, wherein the cells are cancer cells. 14. Use of an antibody of Claim 1 to prepare a medicine for cancer treatment. 15. Use according to Claim 14, wherein the medication induces apoptosis. 16. A pharmaceutical composition consisting of an amount of an antibody of Claim 1 sufficient to induce apoptosis in admixture with a pharmaceutically adjuvant acceptable. 17. The composition of Claim 16, wherein The antibody is a monoclonal antibody. 18. The composition of Claim 16, wherein The antibody is a humanized antibody. 19. The composition of Claim 16, wherein The antibody is a human antibody.